Plasmalogens are a subclass of glycerophospholipid that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in a cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with approximately 730 mU mg−1 protein of specific activity, and the yield was approximately 420 mU l−1 culture corresponding to 76 mU per gram wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.

Plasmalogens, functional glycerophospholipids with biological roles in the human body, are associated with various diseases. Although a variety of saturated and/or unsaturated fatty acids in plasmalogens are presumed to have different functions in the human body, there are limited reports validating such functions of plasmalogens. In this study, we focused on the bacterial plasmalogen derived from Selenomonas ruminantium subsp. lactilytica (NBRC No. 103574) with different main species of hydrocarbon chains at the sn-1 position and shorter fatty acids at the sn-2 position than animal plasmalogens. Optimum culture conditions of S. ruminantium for high-yield production of plasmalogens, such as pH and the concentration of caproic acid, were investigated under anaerobic conditions using a 2-L scale jar fermenter. The obtained plasmalogen mainly consisted of the ethanolamine plasmalogen (PlsEtn). The molar ratios of PlsEtn species obtained from S. ruminantium, at sn-1/sn-2 positions, were p16:1/14:0 (68.4%), p16:1/16:1 (29.2%), p16:1/16:0 (0.7%), p16:1/15:0 (0.3%), and p17:1/14:0 (0.3%). Subsequently, duodenal infusion of the emulsion carrying the lipid extracted from S. ruminantium was carried out in lymph duct-cannulated rats. In the lymphatic plasmalogen of rats, the level of PlsEtns with molar ratios p16:1/14:0 and p16:1/16:1, the main species of plasmalogens from S. ruminantium, increased gradually until 3–4 h after lipid injection and then gradually decreased. In addition, the level of PlsEtns with p16:1/20:4 and p16:1/22:6 rapidly increased, peaking at 1–1.5 h and 1.5–2 h after lipid injection, respectively. The increase in the number of PlsEtns with p16:1/20:4 and p16:1/22:6 suggested that 20:4 and 22:6, the main fatty acids at the sn-2 position in the rat lymphatic plasmalogen, were preferentially re-esterified at the sn-2 position, regardless of the types of hydrocarbon chains at the sn-1 position. Thus, we showed that bacterial PlsEtns with “unnatural” structures against rats could be absorbed into the lymph. Our findings provide insights into the association between the chemical structure of plasmalogens and their biological functions in humans.